Despite recent advance in antibiotic therapy and intensive care, sepsis remains the most common cause of death in the intensive care unit, claiming > 225,000 victims annually in the U.S. alone. Its pathogenesis remains poorly understood, but is partly attributable to dysregulated inflammatory responses that are propagated by early cytokines (e.g., TNF and IFN-?) and sustained by late-acting mediators (e.g., HMGB1). Agents targeting early cytokines (e.g., TNF) could be protective if given prophylactically; whereas agents capable of inhibiting HMGB1 release could rescue animals from lethal sepsis even if given after the onset of sepsis. Our seminal discovery of HMGB1 as a late mediator of lethal sepsis has prompted further investigation of the mechanisms underlying the regulation of its release. On one hand, early cytokines (e.g., TNF, IL-1, IL-6, or IFN-?) directly stimulate innate immune cells (e.g., macrophages and monocytes) to release HMGB1, thereby contributing to endotoxin-induced HMGB1 release. On the other hand, they alter the expression of liver-derived acute phase proteins (APPs), which may similarly participate in the regulation of HMGB1 release. We have generated exciting preliminary data indicating that human serum amyloid A (SAA), but not SAA1, effectively induced HMGB1 release in vitro, and exacerbated endotoxin-mediated animal lethality in vivo. In contrast, SAA-specific neutralizing antibodies and peptides antagonists attenuated SAA-induced HMGB1 release, and conferred protection in animal models of lethal endotoxemia and sepsis. These exciting findings raised several important questions regarding the novel mechanisms underlying the regulation of SAA-mediated HMGB1 release, as well as the possible roles of SAA in the pathogenesis of lethal systemic inflammation (LSI). The experiments outlined in Aim 1 will test the hypothesis that SAA functions as a positive regulator of endotoxin-induced HMGB1 release through TLR4/RAGE receptors. Specifically, we will determine whether inhibition of SAA expression (by gene knockout) or activities (by using neutralizing antibodies, HDL, or peptide antagonists) will affect LPS-, TSST-1-, IFN-?-, or SAA-induced HMGB1 release in wild-type, and SAA- or SAA receptor (e.g., CD36, TLR2, TLR4, or RAGE)-deficient macrophages. In Aim 2, we will test the hypothesis that SAA contributes to the pathogenesis of lethal systemic inflammation by examining whether alteration of SAA levels (by gene knockout or protein supplementation) or activities (by using neutralizing antibodies, HDL, or peptide antagonists) influences animal survival rates in endotoxemia or sepsis. The experiments outlined in Aim 3 will test the hypothesis that that SAA or inhibitors affect LSI through altering systemic cytokine (e.g., HMGB1, sPLA2, or others) accumulation, leukocyte infiltration and bacterial clearance, or hepatic injury from excessive autophagy or amyloidosis. The completion of these exciting studies will further improve our understanding of the mechanisms underlying the regulation of HMGB1 release, and provide guidance for future development of therapeutic strategies to treat lethal inflammatory diseases.